The 5' cap structure (m7GpppN) of eukaryotic messenger RNA modulates the function and metabolism of RNA in the cell. The cap structure is directly involved in RNA function through recognition of mature mRNA by the translation machinery. The cap structure also protects mRNA from degradation. Capping occurs through a series of three chemical reactions. The first reaction is catalyzed by RNA triphosphatase, which removes the terminal gamma phosphate from the nascent 5' triphosphate terminated mRNA chain. This reaction leaves a 5' diphosphate RNA end that is subsequently capped with GMT in a second reaction catalyzed by RNA guanylyltransferase. The third reaction in the capping process is mediated by RNA (guanine-7) methyltransferase, which methylates the N7 position of the terminal guanine. Disruption of any of these catalytic steps blocks cell growth. The main objectives of this proposal are to illuminate the structural basis for eukaryotic mRNA capping by determining crystal structures for several cap forming enzymes in complex with one another, and in complex with key nucleotide and RNA catalytic intermediates. A focus of this research will be to devise methods for determining crystal structures of unique RNA catalytic intermediates that will provide insights into RNA recognition and catalytic mechanism. Another focus will be to structurally characterize the multimeric macromolecular complexes necessary for RNA capping in the cell. We are specifically targeting structure determinations in two fungal systems because these organisms utilize specific mechanisms unique to both viral and fungal pathogens, thus making them ideal targets for structure-based drug design. Structure-based approaches to disrupting the capping process in these organisms will further the development of therapeutic intervention against them.